Abstract Chronic orofacial neuropathic pain that persists after motor vehicle accidents and other facial injuries can be excruciating, unrelenting, and a difficult clinical challenge. Sensitized nerves in the face overactivate not only the trigeminal ganglia neurons but also the second order trigeminal spinal V dorsal horn neurons and glia. Continued activation of pain transmission circuitry results in central sensitization and can exert powerful dysfunctional influences at brain sites associated with anxiety and PTSD. Chronic pain can induce permanent brain circuitry alterations that further diminish physical and mental function. Current treatment with analgesics combined with antidepressants and/or anticonvulsants are generally unsatisfactory in providing pain relief. While opiates are heavily overprescribed to the point of national crisis, they are of little therapeutic value for treatment of orofacial pain. Effective non-addictive, non-opioid therapeutics for chronic orofacial pain remain a critical need. Experimental models in which pain signaling pathways are chronically activated by mild traumatic injury to the trigeminal nerve offer a compelling avenue for discovery and development of new non-addictive, non-opioid therapies. In our trigeminal neuropathic orofacial pain mouse model the trigeminal nerve is compressed to mimic post-traumatic orofacial neuropathic pain in patients. Our recent gene profiling revealed significant 4.0- and 2.7- fold increases in cholecystokinin B (CCK-B) receptor gene expression in the trigeminal ganglia at 3 and 21 days, respectively. The proposed collaborative, multidisciplinary studies will develop novel, brain-penetrant single- chain Fragment variable (scFv) antibody therapies to block CCK-B receptor activation. Aim 1 proposes to generate a repertoire of scFv antibodies using cell-free ribosome display technology to develop, characterize, and authenticate a panel of antibodies to block CCK-B receptor activation. The Aim 2 studies propose testing optimal CCK-B receptor scFv antibodies as therapy to alleviate reflexive and cognitive dependent pain- and anxiety-related behaviors in our orofacial pain model. Preliminary data demonstrate the effectiveness of a single dose of CCK-B receptor scFv antibody administered 3 weeks after model induction during the transition from acute to chronic pain. The proposed studies will further test efficacy of scFv antibody therapy at 8 weeks when hypersensitivity is chronic. Addition of a fluorescent tag on the scFv antibodies will allow additional experimental read-outs including biodistribution with in vivo imaging and postmortem neuropathology to characterize the neuronal and glial cell types involved and their localization. Hypothesis: scFv antibody block of CCK-B receptors is effective therapy for chronic orofacial pain. Aim 1. Generate a panel of CCK-B receptor-specific single-chain Fragment variable (scFv) antibodies using cell-free ribosome display technology to develop/characterize/authenticate a pain therapeutic. Aim 2. Evaluate the effectiveness of CCK-B receptor scFv antibody therapy in male and female mice with acute to chronic and chronic trigeminal nerve injury-induced pain- and anxiety-related behaviors.